Journal: Journal of Cellular and Molecular Medicine

Article Title: The sinus venosus myocardium contributes to the atrioventricular canal: potential role during atrioventricular node development?

doi: 10.1111/jcmm.12525

Figure Lengend Snippet: qPCR primers

Article Snippet: Sections were incubated with primary antibodies against ISL1 (Clone 40.2d6, 1/100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), Troponin I, isoform 2 (TNNI2) (SC-15368, 1/200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), NKX2-5 (SC-8697, 1/500; Santa Cruz Biotechnology Inc.) and HCN4 (APC-052, 1/2000; Alomone Labs, Jerusalem, Israel) overnight.

Techniques: Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: The sinus venosus myocardium contributes to the atrioventricular canal: potential role during atrioventricular node development?

doi: 10.1111/jcmm.12525

Figure Lengend Snippet: HCN4 expression in myocardial continuity between sinus venosus and posterior AV canal. (A) TNNI2 stain of HH19 chick embryo. Arrow indicates continuity between sinus venosus myocardium and posterior AV canal. (B) ISH shows HCN4 mRNA expression in continuity (arrow). (C) Merge of ISL1, TNNI2, HCN4 and DAPI staining of E11.5 mouse embryo, showing the myocardial continuity between the sinus venosus and posterior AV canal. The ISL1+/HCN4+/TNNI2+ SAN (arrowhead) and RVV (arrow) are shown. Note the thick endocardial cushions (asterisks) in the AVC. (D–F) Higher magnifications of boxed area in C and D. ISL1+/TNNI2+/HCN4+ cells are seen in the continuity between sinus venosus myocardium and posterior AV canal myocardium. (E and F) Grey values of ISL1 (E) and HCN4 (F). AVC: atrioventricular canal; LA: left atrium; LB: long bud; LCV: left cardinal vein; LV: left ventricle; OFT: outflow tract; RA: right atrium; RCV: right cardinal vein; SV: sinus venosus; V: ventricle. (C–F) White: TNNI2, green: ISL1, red: HCN4, blue: DAPI; scale bars: 50 μm.

Article Snippet: Sections were incubated with primary antibodies against ISL1 (Clone 40.2d6, 1/100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), Troponin I, isoform 2 (TNNI2) (SC-15368, 1/200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), NKX2-5 (SC-8697, 1/500; Santa Cruz Biotechnology Inc.) and HCN4 (APC-052, 1/2000; Alomone Labs, Jerusalem, Israel) overnight.

Techniques: Expressing, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: The sinus venosus myocardium contributes to the atrioventricular canal: potential role during atrioventricular node development?

doi: 10.1111/jcmm.12525

Figure Lengend Snippet: Characterization of the myocardial continuity between the sinus venosus and AV canal. (A) Whole mount ISL1 staining, posterior view of the heart. Arrows indicate ISL1+ cells in continuity sinus venosus and AV canal. Rectangle 1 shows region from which SV-AVC continuity cells were collected. Rectangle 2 shows origin of right ventricular cells for patch clamp. (B–E) mRNA expression after laser capture microscopy (LCM) of TNNT2, NKX2-5, HCN4 and ISL1 . (F–M) Results of single cell patch-clamp experiments. (F and J) Representative tracing of SV-AVC continuity cells (F, pacemaker-like tracing) and RV cells (J, ventricular-like phenotype). (G–I, K–M) Graphs showing the difference in maximum upstroke velocity (G), frequency (H), amplitude (I), APD50 (K), APD90 (L) and maximum diastolic potential (M). * Indicates P < 0.05. AVC: AV canal; LCM: laser capture microscopy; LV: left ventricle; OFT: outflow tract; RV: right ventricle; SV: sinus venosus; SV-AVC: myocardial continuity sinus venosus and AV canal; scale bar: 250 μm.

Article Snippet: Sections were incubated with primary antibodies against ISL1 (Clone 40.2d6, 1/100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), Troponin I, isoform 2 (TNNI2) (SC-15368, 1/200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), NKX2-5 (SC-8697, 1/500; Santa Cruz Biotechnology Inc.) and HCN4 (APC-052, 1/2000; Alomone Labs, Jerusalem, Israel) overnight.

Techniques: Staining, Patch Clamp, Expressing, Microscopy

Journal: Sleep

Article Title: Altered sleep architecture, rapid eye movement sleep, and neural oscillation in a mouse model of human chromosome 16p11.2 microdeletion

doi: 10.1093/sleep/zsy253

Figure Lengend Snippet: Intrinsic membrane properties and current–voltage (I–V) relationship of vlPAG-projecting GABAergic neurons in LPGi of ventral medulla. (A) Schematic diagram highlighting the ventral lateral periaqueductal gray (vlPAG). (B) A representative fluorescent image showing bilateral vlPAG injected with CTB-488. (C) Schematic diagram of medulla demonstrating LPGi in ventral medulla (reference needed). Laterodorsal tegmental nucleus (LDT) and dorsal raphe nucleus (DRN) are shown as reference. (D) A representative fluorescent image illustrating unilateral LPGi labeled with retrograde CTB-488. The gigantocellular reticular nucleus (Gi), inferior olivary (IO) nucleus, and the nucleus of ambiguus (Amb) are shown for reference. (E) A representative fluorescent image showing a CTB-488 labeled LPGi neuron under patch clamp recording. (F) Post-recording detection of a patched neuron (filled with biocytin via patch pipette) with AMCA-conjugated avidin D. Notice that well-circumscribed contour of the neuron in contrast to irregular background staining. (G) Post-recording verification of Cre expression in the same patched neuron as in (F). Notice that multiple neurons are immunoreactive for Cre. (H) A merged image of (F) and (G). Colocalization of AMCA signal and Cre expression confirmed that the patched neuron is indeed GABAergic. (I) Resting membrane potential (RMP) is mildly reduced in mutant LPGi neurons. (J) Membrane resistance (Rm) is increased in mutant LPGi neurons. (K) Membrane capacitance (Cm) is indistinguishable between wildtype and mutant LPGi neurons. (L) Representative traces of current-voltage relationship in wildtype and mutant LPGi neurons. Both peak (denoted by arrows) and steady-state responses (the last 100 ms denoted by horizontal bars) are subjected to analysis. No statistical differences are present between wildtype and mutant LPGi neurons in peak current (M) and steady state (N) current responses. Schematic diagrams in panels A and C are from Paxinos and Frankin [46]. Arrows and insets in (E) to (H) denote and highlight a patched neuron. Scale bars are 200 microns in (A) to (D), and 30 microns in (E) to (H). Eighteen neurons from 6 wildtype mice and 15 neurons from 5 mutant mice were analyzed. Statistical significance was determined by two-tailed, unpaired Student’s t-test. ns: not significant, *p < 0.05, ***p < 0.001.

Article Snippet: Slices were first washed with 1× PBS supplemented with 3% Triton X (PBST) for three times with 5 min each; they were incubated in 1× PBST + 5% normal donkey serum for 1 h; they were then incubated with AMCA Avidin D (dilution: 1/200; Vector Laboratories, Cat# A-2008) and monoclonal rabbit anti-Cre antibody (dilution: 1/200; Synaptic System, Cat# 257003) at 4°C.

Techniques: Injection, Labeling, Patch Clamp, Transferring, Avidin-Biotin Assay, Staining, Expressing, Mutagenesis, Two Tailed Test

Journal: Molecular Psychiatry

Article Title: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior

doi: 10.1038/s41380-018-0260-9

Figure Lengend Snippet: Lack of SERT increases the number of functional PFC-to-DRN synapses. a rAAV-CAG-hChR2(H134R)-mCherry was bilaterally injected into the PFC of P4–P5 control or SERT-KO mice. Photograph showing mCherry expression after the PFC AAV injection (upper left). Optogenetic stimulation and electrophysiological patch clamp recordings were made starting at P28 in coronal sections containing the DRN, as shown by the photograph of the immunolabeling of PFC mCherry+ axons innervating to DRN 5-HT neurons, identified by the presence of the enzyme TPH2 (upper right). b Amplitude of optogenetically evoked EPSCs (oEPSCs) at synapses from PFC terminals onto DRN putative 5-HT neurons (left) and non-5-HT neurons (right) at various light stimulation intensities. In control (SERT Cre/+ ) (5-HT: n = 10 cells/5 animals; non-5-HT: n = 7 cells/4 animals); in SERT-KO (SERT Cre/Cre ) (5-HT: n = 10 cells/3 animals; non-5-HT: n = 6 cells/3 animals). Top: example traces at 9.8 mW (black/gray) and at 2 mW (red) stimulation); Bottom: input/output curves. Two-way ANOVA on 9.8 mW intensity: genotype x cell-type interaction (F 1,29 = 0.003, p = 0.95); Genotype main effect (F 1,29 = 9.32, * p < 0.01); Cell-type main effect (F 1,29 = 0.51, p = 0.48). c AMPAR/NMDAR ratios at synapses from PFC-to-DRN 5-HT neurons (left) and non-5-HT neurons (right) in control (5-HT: n = 10 cells/4 animals; non-5-HT: n = 7 cells/3 animals), and SERT-KO (5-HT: n = 11 cells/3 animals; non-5-HT: n = 6 cells; 3 animals). The AMPAR responses were calculated at the peak of −50 mV, whereas NMDAR responses were determined at + 40 mV, 50 ms after stimulation. Top: example traces; bottom: bar graphs. Two-ways ANOVA: Genotype x Cell-type interaction (F 1,30 = 0.007, p = 0.94); Genotype main effect (F 1,30 = 0.16, p = 0.69); Cell-type main effect (F 1,30 = 4.51, p < 0.05). Blue bars indicate blue light stimulation. Error bars represent SEM

Article Snippet: Subsequently, after a few hours of fixation in 4% PFA at 4 °C, brain slices were processed for immunohistochemistry using a rabbit polyclonal antibody against TPH2 (1:2000, Novus Biologicals, NB100-74555) to identify 5-HT neurons.

Techniques: Functional Assay, Injection, Control, Expressing, Patch Clamp, Immunolabeling

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin fragments are differentially localized in neurons. ( A ) WB of mouse whole brain protein samples with menin C-terminal ((C); left panel) and N-terminal ((N); right panel) epitope antibodies (n = 3 each, representative blots), depicting full length menin (black arrow), as well as N-terminal (light grey arrow) and C-terminal (dark grey arrow) menin proteolytic fragments. ( B ) WB of subcellular fractions from mouse brain protein samples with menin C-terminal ((C); top and middle panels) and N-terminal ((N); bottom panel) epitope antibodies (n = 6, representative blots). N denotes nuclear fraction, C denotes cytoplasmic fraction, S denotes synaptic fraction. Menin localizes to the nucleus, the C-menin fragment localizes to synaptic membranes, and the N-menin fragment localizes to the cytoplasm. ( C ) As in ( B ), the markers histone H3 (HH3; nuclear marker, top panel), β-tubulin (TUB; cytoplasmic marker, middle panel), and synaptophysin (SYP; synaptic marker, bottom panel), are shown to verify the subcellular fractions. ( D ) ICC localization of menin in hippocampal cultures at DIV 7 (n = 13 images, 4 independent samples, representative image), with N-terminal (i) and C-terminal (ii) epitope antibodies, and the nuclear stain DAPI (iii), (iv) shows merged channels. α-N-terminal menin signals are restricted to the nuclear and perinuclear region, α-C-terminal menin signals also exhibit punctate localization along neurites. Scale bar, 50 μm. See also Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Marker, Staining

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin is cleaved by calpain. ( A ) ICC localization of menin in hippocampal cultures at DIV 7, cultured in the presence of 0.1% DMSO vehicle control (i–v) or 20 μM PD150606 (vi–x), a cell permeable calpain inhibitor (n = 9 images, 3 independent samples each, representative images). Cells were labeled with the nuclear stain DAPI (i,vi), menin N-terminal (ii,vii) and C-terminal (iii,viii) epitope antibodies, and a neurofilament antibody (iv,ix), (v,x) shows merged channels. Scale bar, 50 μm. ( B ) Enlarged region of interest (ROI) depicting neurite structure from the boxed region shown in Av (i–iii) and Ax (iv–vi). Scale bar, 10 μm. ( C , D ) Summary data, fluorescence intensity of the nuclear α-N-menin signal was unaffected ( C ; ROIs: 0.1% DMSO n = 67, 20 μM PD150606 n = 59), whereas the neurite α-C-menin signal was reduced upon calpain inhibition ( D ; ROIs: n = 27 each). ***Statistical significance (independent t-test), P < 0.001. See also Table and Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Cell Culture, Control, Labeling, Staining, Fluorescence, Inhibition

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: The C-terminal menin fragment colocalizes with α7 subunit-containing nAChRs at presynaptic terminals. ( A ) Super resolution image of a synaptic ROI at DIV 7 (n = 9 images, 2 independent samples, representative image), cells were labeled with α-C-terminal menin (i), α-bungarotoxin (ii; α7-nAChR), and α-synaptotagmin (iii; presynaptic marker), (iv) shows merged channels. ( B ) As in ( A ), only labeled with α-PSD-95 (iii; postsynaptic marker) (n = 10 images, 2 independent samples, representative image). Arrowheads, extrasynaptic colocalization of C-menin and α7-nAChRs; arrows, synaptic colocalization with synaptotagmin ( A ) or PSD-95 ( B ). Scale bar, 2 μm. ( C ) Summary data, incidence of colocalization of synaptotagmin (SYT; presynaptic), PSD-95 (postsynaptic), and α-bungarotoxin (BTX; α7-nAChR) puncta with C-menin. Asterisks, statistical significance (Kruskal-Wallis test); ** P < 0.01. *** P < 0.001. See also Table and Fig. .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Labeling, Marker

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin mediates subunit-specific transcriptional regulation of nAChR α5. ( A ) Live cell phase contrast ( i-iii ) and GFP fluorescence ( iv-vi ) images of untreated control (i,iv), NTC shRNA (ii,v), and MEN1 shRNA (iii,vi) lentivirus transduced hippocampal cultures at DIV 7 (n = 18 images, 6 independent samples each, representative images). Scale bar, 100 μm. ( B ) Summary data, fold change gene expression in hippocampal cultures at DIV 7, relative to untreated control, determined by qPCR (n = 6, 2 independent experiments each, triplicate replicates). MEN1 knockdown reduces nAChR α5 expression. ND, qPCR signal not detected. *Statistical significance (pair wise fixed reallocation randomization test), P < 0.05–0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Fluorescence, Control, shRNA, Gene Expression, Knockdown, Expressing

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nAChR α5 subunit expression. ( A ) ICC characterization of menin and nAChR protein expression in neuronal soma from NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures (n ≥ 4 images, ≥2 independent samples; see Table ; representative images, DIV 7). Untransduced neurons were GFP negative (−) and transduced neurons were GFP positive (+). The expression of menin was determined with C-terminal (i,v) and N-terminal (ii,vi) epitope antibodies, and nAChRs with a nAChR α5 antibody (iii,vii), or fluorophore-tagged α-BTX (iv,viii; α7-nAChR). ICC labels are shown in a (left panels), and GFP fluorescence is shown in b (right panels). Scale bars, 20 μm. ( B–E ) Summary data, normalized somal fluorescence intensity of α-C-terminal menin ( B ), α-N-terminal menin ( C ), α-nAChR α5 ( D ), and α-BTX ( E ), in GFP + neurons relative to GFP- neurons at DIV 3–14 (ROIs: n ≥ 12 each; see Table ). Dashed lines represent a 1:1 ratio indicating no change. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Expressing, shRNA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: C-menin mediates neurite localization of α7 subunit-containing nAChRs. ( A , B ) ICC characterization of the neurite localization of C-menin and α7-nAChRs in NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures ( A ; n = 18 images, 5 independent samples each; representative images, DIV 7) or hippocampal cultures maintained in the presence of 0.1% DMSO or 20 μM PD150606 ( B ; n = 12 images, 2 independent samples each; representative images, DIV 7). Cells were labeled with the menin C-terminal epitope antibody (i-ii-a) or fluorophore-tagged α-BTX (iii-iv-a), and neurites were visualized with GFP ( A i-iv-b) or neurofilament ( B i-iv-b) antibodies, (i-iv-c) shows merged channels. Scale bars, 20 μm. ( C , D ) Summary data, the α-C-menin signal in neurites was reduced upon MEN1 knockdown ( C ), which coincides with a reduction of the neurite α-BTX signal ( D ). ( E ) Summary data, reduction of C-menin abundance by calpain inhibition (see Fig. ) coincides with a reduction of the neurite α-BTX signal (ROIs: n ≥ 36 each; see Table ), indicating that the synaptic targeting of α7-nAChRs requires the menin C-terminal fragment. Asterisks, statistical significance (Mann-Whitney U test); * P < 0.05. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: shRNA, Labeling, Knockdown, Inhibition, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces the presynaptic clustering of α7 subunit-containing nAChRs. ( A , B ) Super resolution images of C-menin, α-BTX, and SYT puncta in GFP+ synaptic ROIs from NTC shRNA ( A ) and MEN1 shRNA ( B ) lentivirus transduced hippocampal cultures at DIV 7 (n ≥ 10 images, 2 independent samples each, representative images). Cells were labeled with C-terminal menin and SYT antibodies (i–v; NTC: n = 16, MEN1 : n = 14), fluorophore-tagged α-BTX and a SYT antibody (vi–x; NTC: n = 16, MEN1 : n = 15), or α-BTX and a C-terminal menin antibody (xi–xv; NTC: n = 11, MEN1 : n = 10). (i,vi,xi) shows GFP fluorescence, (ii,xiii) shows α-C-terminal menin fluorescence, (vii,xii) shows α-BTX fluorescence, (iii,viii) shows α-SYT fluorescence, (iv,ix,xiv) shows SYT/C-menin/α-BTX merged channels, (v,x,xv) shows channels merged with GFP. Scale bar, 2 μm. ( C ) Summary data, mean number of α-C-terminal menin, α-BTX, and α-SYT puncta in super resolution images. ( D ) Summary data, incidence of colocalization of SYT/C-menin/α-BTX puncta in super resolution images. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, shRNA, Labeling, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nicotine-induced presynaptic facilitation. ( A , B ) Representative patch clamp traces recorded from GFP+ hippocampal pyramidal neurons at DIV 10–14 (n ≥ 15, 3 independent experiments, representative traces). The mean frequency and amplitude of mEPSCs was analyzed before (Pre-nicotine, 10 s; top trace) and after (Post-nicotine, 10 s; bottom trace) the nicotine pulse (10 μM, 250 ms, 10 PSI; not shown). nAChR-mediated increase in mEPSC frequency (e.g. arrow) is observed in a GFP+ neuron from NTC-shRNA expressing cultures ( A ), but is absent in a GFP+ neuron from MEN1 -shRNA expressing cultures ( B ). ( C ) Summary data, incidence of nicotine-induced presynaptic facilitation in neurons from untreated control (n = 13/19), NTC shRNA (n = 11/15; GFP+) and MEN1 shRNA (n = 5/17; GFP+) transduced hippocampal cultures (post/pre-nicotine relative mEPSC frequency ≥1 ± SEM; see Fig. and Table ). *Statistical significance (Chi-squared test), P < 0.05. ( D ) Summary data, mean frequency of mEPSCs before (pre) and after (post) the nicotine pulse in the population of neurons from untreated control (n = 13), NTC shRNA (n = 11; GFP+) and MEN1 shRNA (n = 5; GFP+) transduced cultures that exhibit nicotine-induced presynaptic facilitation. ***Statistical significance (paired t-test) , P < 0.001. ( E ) Summary data, mean amplitude of mEPSCs, as in ( D ). See also Tables – and Figs – .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Patch Clamp, shRNA, Expressing, Control

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin fragments are differentially localized in neurons. ( A ) WB of mouse whole brain protein samples with menin C-terminal ((C); left panel) and N-terminal ((N); right panel) epitope antibodies (n = 3 each, representative blots), depicting full length menin (black arrow), as well as N-terminal (light grey arrow) and C-terminal (dark grey arrow) menin proteolytic fragments. ( B ) WB of subcellular fractions from mouse brain protein samples with menin C-terminal ((C); top and middle panels) and N-terminal ((N); bottom panel) epitope antibodies (n = 6, representative blots). N denotes nuclear fraction, C denotes cytoplasmic fraction, S denotes synaptic fraction. Menin localizes to the nucleus, the C-menin fragment localizes to synaptic membranes, and the N-menin fragment localizes to the cytoplasm. ( C ) As in ( B ), the markers histone H3 (HH3; nuclear marker, top panel), β-tubulin (TUB; cytoplasmic marker, middle panel), and synaptophysin (SYP; synaptic marker, bottom panel), are shown to verify the subcellular fractions. ( D ) ICC localization of menin in hippocampal cultures at DIV 7 (n = 13 images, 4 independent samples, representative image), with N-terminal (i) and C-terminal (ii) epitope antibodies, and the nuclear stain DAPI (iii), (iv) shows merged channels. α-N-terminal menin signals are restricted to the nuclear and perinuclear region, α-C-terminal menin signals also exhibit punctate localization along neurites. Scale bar, 50 μm. See also Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Marker, Staining

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin is cleaved by calpain. ( A ) ICC localization of menin in hippocampal cultures at DIV 7, cultured in the presence of 0.1% DMSO vehicle control (i–v) or 20 μM PD150606 (vi–x), a cell permeable calpain inhibitor (n = 9 images, 3 independent samples each, representative images). Cells were labeled with the nuclear stain DAPI (i,vi), menin N-terminal (ii,vii) and C-terminal (iii,viii) epitope antibodies, and a neurofilament antibody (iv,ix), (v,x) shows merged channels. Scale bar, 50 μm. ( B ) Enlarged region of interest (ROI) depicting neurite structure from the boxed region shown in Av (i–iii) and Ax (iv–vi). Scale bar, 10 μm. ( C , D ) Summary data, fluorescence intensity of the nuclear α-N-menin signal was unaffected ( C ; ROIs: 0.1% DMSO n = 67, 20 μM PD150606 n = 59), whereas the neurite α-C-menin signal was reduced upon calpain inhibition ( D ; ROIs: n = 27 each). ***Statistical significance (independent t-test), P < 0.001. See also Table and Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Cell Culture, Control, Labeling, Staining, Fluorescence, Inhibition

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: The C-terminal menin fragment colocalizes with α7 subunit-containing nAChRs at presynaptic terminals. ( A ) Super resolution image of a synaptic ROI at DIV 7 (n = 9 images, 2 independent samples, representative image), cells were labeled with α-C-terminal menin (i), α-bungarotoxin (ii; α7-nAChR), and α-synaptotagmin (iii; presynaptic marker), (iv) shows merged channels. ( B ) As in ( A ), only labeled with α-PSD-95 (iii; postsynaptic marker) (n = 10 images, 2 independent samples, representative image). Arrowheads, extrasynaptic colocalization of C-menin and α7-nAChRs; arrows, synaptic colocalization with synaptotagmin ( A ) or PSD-95 ( B ). Scale bar, 2 μm. ( C ) Summary data, incidence of colocalization of synaptotagmin (SYT; presynaptic), PSD-95 (postsynaptic), and α-bungarotoxin (BTX; α7-nAChR) puncta with C-menin. Asterisks, statistical significance (Kruskal-Wallis test); ** P < 0.01. *** P < 0.001. See also Table and Fig. .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Labeling, Marker

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin mediates subunit-specific transcriptional regulation of nAChR α5. ( A ) Live cell phase contrast ( i-iii ) and GFP fluorescence ( iv-vi ) images of untreated control (i,iv), NTC shRNA (ii,v), and MEN1 shRNA (iii,vi) lentivirus transduced hippocampal cultures at DIV 7 (n = 18 images, 6 independent samples each, representative images). Scale bar, 100 μm. ( B ) Summary data, fold change gene expression in hippocampal cultures at DIV 7, relative to untreated control, determined by qPCR (n = 6, 2 independent experiments each, triplicate replicates). MEN1 knockdown reduces nAChR α5 expression. ND, qPCR signal not detected. *Statistical significance (pair wise fixed reallocation randomization test), P < 0.05–0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Fluorescence, Control, shRNA, Gene Expression, Knockdown, Expressing

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nAChR α5 subunit expression. ( A ) ICC characterization of menin and nAChR protein expression in neuronal soma from NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures (n ≥ 4 images, ≥2 independent samples; see Table ; representative images, DIV 7). Untransduced neurons were GFP negative (−) and transduced neurons were GFP positive (+). The expression of menin was determined with C-terminal (i,v) and N-terminal (ii,vi) epitope antibodies, and nAChRs with a nAChR α5 antibody (iii,vii), or fluorophore-tagged α-BTX (iv,viii; α7-nAChR). ICC labels are shown in a (left panels), and GFP fluorescence is shown in b (right panels). Scale bars, 20 μm. ( B–E ) Summary data, normalized somal fluorescence intensity of α-C-terminal menin ( B ), α-N-terminal menin ( C ), α-nAChR α5 ( D ), and α-BTX ( E ), in GFP + neurons relative to GFP- neurons at DIV 3–14 (ROIs: n ≥ 12 each; see Table ). Dashed lines represent a 1:1 ratio indicating no change. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Expressing, shRNA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: C-menin mediates neurite localization of α7 subunit-containing nAChRs. ( A , B ) ICC characterization of the neurite localization of C-menin and α7-nAChRs in NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures ( A ; n = 18 images, 5 independent samples each; representative images, DIV 7) or hippocampal cultures maintained in the presence of 0.1% DMSO or 20 μM PD150606 ( B ; n = 12 images, 2 independent samples each; representative images, DIV 7). Cells were labeled with the menin C-terminal epitope antibody (i-ii-a) or fluorophore-tagged α-BTX (iii-iv-a), and neurites were visualized with GFP ( A i-iv-b) or neurofilament ( B i-iv-b) antibodies, (i-iv-c) shows merged channels. Scale bars, 20 μm. ( C , D ) Summary data, the α-C-menin signal in neurites was reduced upon MEN1 knockdown ( C ), which coincides with a reduction of the neurite α-BTX signal ( D ). ( E ) Summary data, reduction of C-menin abundance by calpain inhibition (see Fig. ) coincides with a reduction of the neurite α-BTX signal (ROIs: n ≥ 36 each; see Table ), indicating that the synaptic targeting of α7-nAChRs requires the menin C-terminal fragment. Asterisks, statistical significance (Mann-Whitney U test); * P < 0.05. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: shRNA, Labeling, Knockdown, Inhibition, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces the presynaptic clustering of α7 subunit-containing nAChRs. ( A , B ) Super resolution images of C-menin, α-BTX, and SYT puncta in GFP+ synaptic ROIs from NTC shRNA ( A ) and MEN1 shRNA ( B ) lentivirus transduced hippocampal cultures at DIV 7 (n ≥ 10 images, 2 independent samples each, representative images). Cells were labeled with C-terminal menin and SYT antibodies (i–v; NTC: n = 16, MEN1 : n = 14), fluorophore-tagged α-BTX and a SYT antibody (vi–x; NTC: n = 16, MEN1 : n = 15), or α-BTX and a C-terminal menin antibody (xi–xv; NTC: n = 11, MEN1 : n = 10). (i,vi,xi) shows GFP fluorescence, (ii,xiii) shows α-C-terminal menin fluorescence, (vii,xii) shows α-BTX fluorescence, (iii,viii) shows α-SYT fluorescence, (iv,ix,xiv) shows SYT/C-menin/α-BTX merged channels, (v,x,xv) shows channels merged with GFP. Scale bar, 2 μm. ( C ) Summary data, mean number of α-C-terminal menin, α-BTX, and α-SYT puncta in super resolution images. ( D ) Summary data, incidence of colocalization of SYT/C-menin/α-BTX puncta in super resolution images. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, shRNA, Labeling, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nicotine-induced presynaptic facilitation. ( A , B ) Representative patch clamp traces recorded from GFP+ hippocampal pyramidal neurons at DIV 10–14 (n ≥ 15, 3 independent experiments, representative traces). The mean frequency and amplitude of mEPSCs was analyzed before (Pre-nicotine, 10 s; top trace) and after (Post-nicotine, 10 s; bottom trace) the nicotine pulse (10 μM, 250 ms, 10 PSI; not shown). nAChR-mediated increase in mEPSC frequency (e.g. arrow) is observed in a GFP+ neuron from NTC-shRNA expressing cultures ( A ), but is absent in a GFP+ neuron from MEN1 -shRNA expressing cultures ( B ). ( C ) Summary data, incidence of nicotine-induced presynaptic facilitation in neurons from untreated control (n = 13/19), NTC shRNA (n = 11/15; GFP+) and MEN1 shRNA (n = 5/17; GFP+) transduced hippocampal cultures (post/pre-nicotine relative mEPSC frequency ≥1 ± SEM; see Fig. and Table ). *Statistical significance (Chi-squared test), P < 0.05. ( D ) Summary data, mean frequency of mEPSCs before (pre) and after (post) the nicotine pulse in the population of neurons from untreated control (n = 13), NTC shRNA (n = 11; GFP+) and MEN1 shRNA (n = 5; GFP+) transduced cultures that exhibit nicotine-induced presynaptic facilitation. ***Statistical significance (paired t-test) , P < 0.001. ( E ) Summary data, mean amplitude of mEPSCs, as in ( D ). See also Tables – and Figs – .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Patch Clamp, shRNA, Expressing, Control

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: Fxyd6 is expressed in small DRG neurons. (a) Different genes have distinct expression patterns in the mouse DRG neurons. Based on the single-cell RNA-seq data, DRG neurons could be classified into 10 types, namely C1-C10. A heatmap showed that each type has its own corresponding markers, such as Gal for C1, C2 Il31ra , C3 Th , C4 Mrgpra3 , and C5 Mrgprd . As demonstrated, Fxyd6 was mainly expressed in small DRG neurons, especially in the C1 Gal + and C3 Th + type neurons. The ranges of the relative expression levels of different genes, and the range of IB4-labeling and the diameter of each type of neurons are presented with colors indicated in the last panel. (b) ISH result demonstrates that Fxyd6 is present in about 23.5% of DRG neurons. (c) The statistical results demonstrated that the Fxyd6 mRNA-containing neurons were small-diameter DRG neurons (cross-sectional area of neuron profiles < 600 µm 2 ).

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Expressing, RNA Sequencing, Labeling

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: FXYD6 is expressed in two subpopulations of small DRG neurons. Double fluorescent ISH and immunostaining showed the expression of FXYD6/ Fxyd6 and related markers in DRG neurons. Fxyd6 was mainly co-expressed with peripherin, but not NF200 and IB4 in DRG neurons. FXYD6 was co-expressed with TH and GAL partially. However, Fxyd6 was not co-expressed with Il31ra and Mrgprd . Scale bar = 50 µm.

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Immunostaining, Expressing

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: FXYD6 protein is transported to the afferent fibers in the superficial laminae of the spinal cord. (a) Immunoblotting result showed that FXYD6 was widely expressed in nervous system, including cortex, cerebellum, brain stem, hippocampus, spinal cord, dorsal root and ventral root, DRG and sciatic nerve. (b) ISH result demonstrated that Fxyd6 mRNA was not detected in the superficial laminae of the lumbar 4 and 5 segments of spinal cord. Fxyd6 mRNA was mainly expressed in the central canal of the spinal cord. Scale bar = 200 µm. (c) Double fluorescent immunostaining showed that the expression of FXYD6 in spinal cord. FXYD6 was co-localized with CGRP and SP, but not IB4 or PKCγ, in the superficial dorsal horn of lumbar spinal cord. Scale bar = 100 µm.

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Western Blot, Immunostaining, Expressing

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: Fxyd6 cKO mice shows impaired behavioral response to noxious heat. (a) The generation strategy of Fxyd6 cKO mice. Double fluorescent ISH showed that Fxyd6 was co-expressed with Na V 1.8 in DRG neurons, so Fxyd6 F/F mice and SNS-Cre mice were crossed to generate Fxyd6 cKO mice. (b) Immunoblotting showed that the FXYD6 level in DRG of Fxyd6 cKO mice was decreased, compared to that of control mice (*** p < 0.001, Fxyd6 cKO mice versus control mice, n = 6). Data shown are mean ± SEM. (c) Hargreaves test showed that the thermal latency of Fxyd6 cKO mice was increased, compared to that of control mice (*** p < 0.001, Fxyd6 cKO mice versus control mice, n = 6). Hotplate test showed that the thermal latency of Fxyd6 cKO mice was increased, compared to that of control mice (* p < 0.05, Fxyd6 cKO mice versus control mice, n = 5). Von Frey test showed that the mechanical threshold of Fxyd6 cKO mice was not significantly altered compared to control mice (n = 6 for both Fxyd6 cKO mice and control mice). Pinprick test showed that the paw withdrawal frequency was unchanged between Fxyd6 cKO mice and control mice (n = 6 for both Fxyd6 cKO mice and control mice). Data shown are mean ± SEM.

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Western Blot, Control

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: FXYD6 co-expresses and interacts with TRPV1. (a) Intraplantar injection of capsaicin or saline into the hindpaw was performed in Fxyd6 cKO mice and control mice. Behavioral responses to intraplantar injection of capsaicin or saline were measured for 10 min after injection. Both Fxyd6 cKO mice and control mice showed little or no responses to the injection of saline ( p = 0.9275, Fxyd6 cKO mice versus control mice, n = 5). When compared to control mice, the licking time after capsaicin injection was decreased in Fxyd6 cKO mice (* p < 0.05, Fxyd6 cKO mice versus control mice, n = 6). Data shown are mean ± SEM. (b) Double-fluorescence immunostaining result showed that FXYD6 and TRPV1 co-expressed in small DRG neurons. About 41.9% FXYD6 + neurons also expressed TRPV1, while about 19.8% TRPV1 + neurons also expressed FXYD6. Scale bar = 50 μm. (c) Co-IP showed that TRPV1 (∼100 kDa) was in the proteins precipitated with the FXYD6 antibody from the mouse DRGs. Three independent experiments were performed with similar results. (d) In the lysate of HEK 293T cells co-transfected with plasmids expressing TRPV1-Flag and FXYD6-Myc, FXYD6-Myc (∼20 kDa) was found in proteins precipitated with Flag antibodies. Three independent experiments were performed with similar results.

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Injection, Saline, Control, Fluorescence, Immunostaining, Co-Immunoprecipitation Assay, Transfection, Expressing

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: FXYD6 increases the capsaicin-sensitive currents. (a) DRG neurons were dissociated acutely and cultured. The patch-clamp recording of these neurons was performed when capsaicin was applied. The currents of DRG neurons responding to capsaicin (5 μM) in Fxyd6 cKO mice were smaller than those in control mice (n = 19 for DRG neurons from control mice and n = 35 for DRG neurons from Fxyd6 cKO mice, *** p < 0.001 Fxyd6 cKO mice versus control mice). Data shown are mean ± SEM. (b) The workflow of electrophysiological studies and single-cell PCR. Small DRG neurons were disassociated acutely, patch-clamped and recorded when capsaicin (5 μM) was applied for 3 times and 10 seconds per time. Then DRG neurons were collected and total RNA were extracted. The cDNA of neurons were obtained by reverse transcription and processed for the single-cell PCR. Finally, all DRG neurons were classified into different types, according to the neuron type markers detected by single-cell PCR. (c) Single-cell PCR showed that 34 DRG neurons were Gal + type neurons, in which 3 neurons didn’t express Trpv1 ( Trpv1 – ) and remaining 31 neurons expressed Trpv1 ( Trpv1 + ). Statistical result showed that TRPV1 capsaicin-sensitive currents in the Trpv1 + /Fxyd6 – Gal + type DRG neurons were smaller, compared to that of Trpv1 + /Fxyd6 + Gal + type of small DRG neurons (n = 17 for Trpv1 + and Fxyd6 + neurons and n = 14 for Trpv1 + but Fxyd6 – neurons, * p < 0.05, unpaired t test). Data shown are mean ± SEM. (d) In HEK293 cells, TRPV1-mCherry was co-transfected with FXYD6-EGFP (TRPV1 + FXYD6) or Vector-EGFP (TRPV1 + Vector). FXYD6 could increase the TRPV1 capsaicin-sensitive currents (n = 26 for TRPV1 + FXYD6 cells and n = 17 for TRPV1 + Vector cells, * p < 0.05, unpaired t test). Data shown are mean ± SEM.

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Cell Culture, Patch Clamp, Control, Reverse Transcription, Transfection, Plasmid Preparation

Journal: Molecular Pain

Article Title: FXYD6 promotes thermal nociception by regulating TRPV1

doi: 10.1177/1744806921992249

Figure Lengend Snippet: The PGDEE motif of FXYD6 is required for the FXYD6/TRPV1 interaction. (a) Alignment of FXYD6 protein sequences of different species indicated the PGDEE motif was completely conserved. (b) In the lysate of HEK 293T cells co-transfected with the plasmids expressing TRPV1-Flag and FXYD6-Myc or FXYD6mut-Myc, Co-IP showed that FXYD6-Myc or FXYD6mut-Myc is present in the proteins precipitated by Flag antibodies. The immunoblot signal of PGDEEmut-Myc was weaker than that of FXYD6 CDS-Myc in cells co-expressing TRPV1-Flag. The immunoblot shown are representatives of four such experiments. (c) Quantitation showed that the interaction between PGDEEmut-Myc and TRPV1-Flag was weaker than that between FXYD6 CDS-Myc and TRPV1-Flag ( t = 7.787, df = 8, *** p < 0.0001, PGDEEmut versus FXYD6 CDS, n = 5; t = 1.666, df = 10, p = 0.1267, PFXYDYmut versus FXYD6 CDS, n = 6; t = 1.745, df = 6, p = 0.1317, (PF+PG)mut versus FXYD6 CDS, n = 4). (d) In HEK293 cells, TRPV1-mCherry was co-transfected with FXYD6 (TRPV1 + FXYD6) or PGDEEmut (TRPV1 + PGDEEmut). The TRPV1 capsaicin-sensitive currents in the PGDEEmut group were significantly decreased when compared to those in the FXYD6 group (n = 5 for both TRPV1 + FXYD6 cells and TRPV1 + PGDEEmut cells, * p < 0.05, unpaired t test). Data shown are mean ± SEM.

Article Snippet: The primary antibodies against FXYD6 (1:2000, Rb, this paper), TRPV1 (1:500, Go, Santa Cruz, sc-12498), TH (1:1000, Sh, Merk/Millipore, AB1542), NF200 (1:2000, Mo, Sigma, N2912), CGRP (1:500, Go, AbD, 1720–9007), Peripherin (1:500, Rb, Chemicon, AB1530), and Nav1.8 (1:1000, Rb, Alomone Labs, ASC-016).

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Quantitation Assay

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Mice were subjected to either intraperitoneal (ip.) or intracortical (cor.) LPS injections, while NKCC1 was blocked by ip. bumetanide (Bum) administration. Central LPS injection triggers high cytokine (G-CSF, IL-1α, IL-1β) and chemokine (KC) responses in the brain compared to ip. LPS injection, which is blocked by ip. Bum administration. B : Central NKCC1 inhibition by intracortical Bum administration significantly increases GCSF and IL-1β levels. See also Supplementary Figure 1 for effects of systemic vs. central blockade of NKCC1 on LPS-induced cytokine responses in the periphery. C : Flow cytometric dot plots show that cortical administration of Bum does not affect the number of microglia (CD45 int /P5 gate), and recruitment of leukocytes (CD45 high /P4 gate), including monocytes (CD11b + , Ly6C high /P9 gate), and granulocytes (CD11b + , Ly6G high /P7 gate) upon central LPS injection. D : The main source of IL-1α and IL-1β in the brain are microglia cells. Confocal images of Cx3CR1 +/GFP brain slices show IL-1α-CD45-P2Y12R (above, red arrowheads) and IL-1β-CD45-P2Y12R (below, red arrowheads) labelled cells after cortical LPS injection-induced inflammation. All data are expressed as mean±SEM. E : NKCC1 (encoded by Slc12a2 ) and P2Y12R gene expression is downregulated in microglia isolated from adult mice 24 hours after cisterna magna LPS application. A : One-way ANOVA followed by Tukey’s multiple comparison test; * p <0.05; N=6/group; B : Unpaired t-test; * p <0.05; N=9/group; Data were pooled from two independent studies. C : One-way ANOVA followed by Tukey’s multiple comparison test; * p <0.05; N=4/group. D : Scale: 25 μm; E : Unpaired t-test; ** p <0.01, *** p <0.001; N (WT)=6, N (KO)=5; Abbreviations: veh.: vehicle; ip: intraperitoneal; cor.: cortical; Bum: bumetanide; ns: not significant

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Injection, Inhibition, Gene Expression, Isolation, Comparison

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A: NKCC1 mRNA expression levels in newborn and adult microglia isolated from C57BL/6J mice compared to neural progenitors derived from E17 embryonic hippocampi. Note, that NKCC1 mRNA levels decrease dramatically during in vitro maintenance (DIV10). B: We generated a novel microglia-specific conditional NKCC1 KO transgenic mouse line by crossing NKCC1 fl/fl (exon 8 of the Slc12a2 gene was flanked with lox P sites) and Cx3CR1-Cre ERT2 mice. C: NKCC1 mRNA levels in isolated NKCC1 KO microglia was markedly reduced in comparison to wild-type cells. D-E: NKCC1 protein expression in a large number of randomly sampled NKCC1 KO microglia cells is markedly reduced compared to WT cells. Inserts show plasma membrane localization of NKCC1. F-G: Automated morphological analysis and maximum intensity projections of confocal images. Inserts show cells marked with white asterisks in 3D. Arrowheads indicate altered branch structure of NKCC1 KO microglia. Automated morphological analysis shows that features of NKCC1 deficient microglia significantly differ from WT microglia. A: One-way ANOVA, followed by Holm-Sidak’s post hoc test. N=3/group. **: p <0.01; n.s.: not significant C: Mann-Whitney test, N=3/group. **: p <0.01 D: Mann-Whitney test, N (WT)=142 cells from 2 mice, N (KO)=83 cells from 1 mouse. ****: p <0.0001 E: Scale: 2 μm F-G: Scale: 10 μm; Mann-Whitney test, N (WT)=78 cells from 3 mice, N (KO)=136 cells from 5 mice. **: p <0.01, ***: p <0.001, ****: p <0.0001 Abbreviations: DIV: days in vitro; n.s.: not significant; TMX: tamoxifen

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Expressing, Isolation, Derivative Assay, In Vitro, Generated, Transgenic Assay, Comparison, Clinical Proteomics, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Baseline NLRP3 and IL-1β mRNA expression is increased in isolated NKCC1 KO microglia compared to WT cells. B: Experimental outline of automated morphological analysis, cytokine array and flow cytometry. C-D : Automated morphological analysis show that activated NKCC1 deficient microglia are slightly smaller than their wild-type counterparts. E : LPS-induced cytokine levels are significantly higher in the cortices of microglial NKCC1 KO mice than in WT. F: Flow cytometric dot plots show that microglial NKCC1 deficiency does not alter the number of CD11b + , CD45 int microglia (P4 gate) or numbers of infiltrating CD11b + , CD45 high leukocytes (P5 gate), CD11b + , Ly6C high monocytes (P9 gate) and CD11b + , Ly6G high granulocytes (P7 gate) in response to intracortical LPS administration. See corresponding data on peripheral cytokine levels and immune cell populations in Supplementary Figure 3 . G: Increased NLRP3 and IL-1β mRNA levels are sustained in NKCC1 KO and WT microglia 24 hours after intracisternal LPS administration . A: Unpaired t-test; * p <0.05, ** p <0.01; N (WT)=6, N (KO)=5 D: Mann-Whitney test, N (WT)=171 cells from 6 mice, N (KO)= 85 cells from 4 mice, ** p <0.01, *** p <0.001 E: Mann-Whitney test, *: p <0.05; N (WT)=7, N (KO)=6 F: unpaired t-test, N (WT)=4, N (KO)=4 G: Unpaired t-test; * p <0.05; N (WT)=5, N (KO)=5; n. s.: not significant.

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Expressing, Isolation, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A : Schematic representation of experiment. Perforated patch-clamp recordings were performed on microglial cells in acute hippocampal slice preparations. Current responses to a train of voltage steps from -100 to 100 mV with 20 mV increments and a duration of 100 ms were measured in voltage-clamp mode (holding potential: -40 mV) both in normotonic, and after 5 minute perfusion with hypotonic ACSF (50% dilution). B: Example traces of recordings from WT (black: normotonic, grey: hypotonic ACSF) and NKCC1 KO (purple: normotonic, violet: hypotonic ACSF) animal. Traces show responses for -100 and +100 mV stimulations in both conditions. C: Average I-V curve responses from WT (black squares with SEM) and NKCC1 KO cells (violet circles with SEM) in normotonic (left) and after 5 minute perfusion of hypotonic ACSF (right) D: Resting membrane potential in normotonic condition of WT vs. NKCC1 KO microglial cells. E: I-V curves calculated by the subtraction of measured values in normotonic conditions from ones in hypotonic medium, resulting in I-V curves representing the currents evoked by cell-swelling due to osmotic change. F: Reversal potentials of the swelling-induced currents measured from WT (grey) or NKCC1 KO (violet) animals (left), with corresponding intracellular Cl - concentrations (right) calculated via the Nernst equation. All parametric data are expressed as mean±SEM. C: Unpaired t-test; N(WT)=8 cells from 7 animal, N(KO)=8 cell from 6 animal; *: p<0.05, **: p<0.01 D: Unpaired t-test; N(WT)=13 microglial cells from 12 mice, N(KO)=12 microglial cells from 11 mice; **: p <0.01 E: Unpaired t-test; *: p <0.05 F: Unpaired t-test; N(WT)=8 cell, N (KO)=8 cell; *: p<0.05.

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Patch Clamp, Membrane

Journal: bioRxiv

Article Title: NKCC1 modulates microglial phenotype, cerebral inflammatory responses and brain injury in a cell-autonomous manner

doi: 10.1101/2021.01.21.427597

Figure Lengend Snippet: A-B : Microglial NKCC1-deficient mice (KO) show larger infarct volume as assessed on cresyl violet-stained brain sections and more severe neurological outcome compared to wild type mice. C: Cytokine levels in the cortex do not differ 8 hours after MCAo in KO mice compared to WT. D-E: Microglial NKCC1 deletion results in higher levels of IL-1α and IL-1β 24 hours after MCAo. B: Unpaired t-test, N (WT)=7, N (KO)=9; *: p <0.05, **: p <0.01 C: Kruskall-Wallis test followed by Dunn’s multiple comparison test; N=6/group. *: p <0.05, **: p <0.01 D: Scale: 50 μm E: Mann-Whitney test, *: p <0.05; N (WT)=7, N (KO)=9. n.s.: not significant

Article Snippet: The diluted anti-NKCC1 primary antibodies (NKCC1 Rb: 1:4000, #13884-1-AP, Proteintech, NKCC1 M: 1:2000, diluted in MOM-diluent; DSHB) were pre-incubated for 48 hours with brain slices from NKCC1 -/- mice, in order to remove the fraction of immunoglobulins that could potentially cause aspecific-binding.

Techniques: Staining, Comparison, MANN-WHITNEY